Our overall goal is to develop a long-term source of human cultured red blood cells (RBCs) from embryonic stem (ES) and induced pluripotent (iPS) cells. More than 16 million units of RBCs are transfused yearly in the US and the need for blood is projected to increase as our population ages. Current reliance on human blood donors as the sole source of RBCs is associated with infectious risks and supply bottlenecks. In the adult, RBCs are derived from erythroid-restricted progenitors that undergo limited ex vivo proliferation when cultured in erythropoietin, stem cell factor, and dexamethasone. We have discovered that erythroid progenitors derived from the yolk sac of mouse embryos and from mouse ES cells can self-renew in vitro more than 10^60-fold, all the while retaining their ability to undergo 3-4 maturational cell divisions to become enucleated RBCs. These extensively self-renewing erythroblasts (ESREs), express adult globin's and produces a wave of fully mature RBCs when injected in vivo. ESREs are derived from erythro-myeloid progenitors (EMP), a transient wave of definitive hematopoiesis that emerges in the yolk sac of mouse and human embryos prior to the appearance of hematopoietic stem cells. We hypothesize that the early ontogeny of the hematopoietic system is conserved in mammalian embryos and that human self-renewing erythroblasts can be generated from the first definitive EMP that emerge in differentiating human ES cells. In Aim 1, we will delineate the emergence of definitive hematopoiesis in differentiating human ES/iPS cells. Importantly, the transduction of Bmi-1 into murine and human erythroblasts dramatically increases their capacity for in vitro self-renewal. In Aim 2, we will apply our knowledge of the mechanisms regulating erythroid self-renewal to optimize the expansion and maturation of human erythroblasts. Completion of these aims will provide a better understanding of erythroblast self-renewal and bring an ever-renewable source of human erythroblasts closer to the clinic to meet the transfusional needs of critically ill children and adults.